Project Summary Fully half of all HIV-infected individuals continue to display some (often milder) form of HIV-associated neurocognitive disorder (HAND) despite the introduction of antiretroviral therapy (ART). More than one in 10 of these individuals will exhibit progressive neurologic deterioration on ART. More severe forms of HAND, including HIV-associated dementia, remain common in the developing world, especially in individuals not receiving ART. HAND is likely caused by chronic inflammation in the brain leading to neuronal dysfunction. The conundrum is how this inflammatory response is sustained despite effective suppression of viral replication with ART. We believe latent HIV infection of microglia likely plays a central role. Microglia comprise 10-15% of all cells in the CNS and serve as the brain's constant gardeners shaping neuronal plasticity through synaptic pruning and stripping; microglia also participate in bidirectional signaling with closely intertwined neurons. How best to study these microglia, their interplay with neurons, and the effects of HIV infection? We propose to coculture two iPSC- derived sub-lines engineered to express doxycycline-inducible transcription factors that are sufficient to drive differentiation into either microglia or excitatory neurons. When induced and cocultured in 3D conditions, these cells form cerebral microorganoids (CMs) that recapitulate many of the cytoarchitectural features and functions of the fetal brain. We will study these CMs in an unbiased manner using scRNA-seq to define gene expression profiles and scATAC-sec to interrogate chromatin accessibility. Use of a combinatorial indexing system of barcodes will allow measurement of these parameters in the same cell. We hypothesize that microglia are latently infected and that sustained neuronal neurotransmitter signaling is likely sufficient to reactivate virus expression plus exposure to opioids will further enhance reactivation (virus production is not impaired by ART). Release of reactivated virions may directly trigger a chronic inflammatory response. Additionally, when these viruses are transmitted cell-to-cell, an abortive form of HIV infection may ensue due to the action of the RT inhibitors present in ART. The IFI16 DNA sensor may detect these RT products leading to inflammasome assembly, caspase-1 activation, production of IL-1? and IL-18 and death by pyroptosis, a highly inflammatory form of programmed cell death. Because pyroptosis breeds more pyroptosis, this feed-forward form of inflammation could a create chronic inflammatory response resistant to ART. Finally, we are eager to explore two CNS-tailored approaches for attacking the latent HIV reservoir in microglia. In the first, virus will be purged with a CNS-penetrant LRA and cells producing viral RNA will be selectively killed by induction of RIG-I-dependent apoptosis. In the second, durable, sequence-specific transcriptional silencing of HIV proviruses will be tested using CRISPR interference to promote H3K9me3 and DNA methylation??both epigenetic modifications are needed for long term silencing. Together, these studies promise to provide new and exciting insights into HAND pathogenesis, HIV latency in the brain, effects of opioids, and the potential link between these processes.